Production methods

ABSTRACT

The present invention provides methods of reducing the levels of a titratable selectable pressure required, the number of amplification cycles, and the time taken to generate protein expressing cell lines by altering the codons of the desired open-reading-frames. Through the use of codon adaptation for this purpose the methods of the invention consistently provide sufficient yields in faster time frames saving many weeks in cell line development activities. Furthermore the methods of the invention also generate cell lines with lower concentrations of selection and amplification agent than previously achievable. Accordingly lower levels of selection and amplification marker in the final cells lines are observed.

The present invention provides a method for producing a cell line whichis capable of secreting a therapeutic protein. The method comprises theuse of codon adapted gene sequences which results both in reducedprotocol timelines and a decrease in the concentrations of antifolaterequired when generating eg. antibody producing cell lines via aselection and amplification system.

Mammalian cells such as CHO (Chinese Hamster Ovarian cells), NSO andPerC6 cells are routinely employed within the biopharmaceutical industryto manufacture biopharmaceuticals. These cells are geneticallyengineered and then selected in such a way as to ensure that high titreexpression of the desired protein is observed when the resulting celllines are cultured in bioreactors.

Currently there are a number of methods to engineer and then select thebest cells for this purpose. Often these methods involve ‘amplification’to increase copy number of the integrated expression vector or vectorsto improve yields observed of the desired protein. These ‘amplification’methods are well described previously by Bebbington and Hentschel (DNACloning Volume III (IRL press, 1987)). The authors explain that a numberof selectable markers (which are often in the form of nucleic acidsequences encoding enzymes involved in metabolism and essential for thehost cells survival under certain culture media conditions) can beoperatively linked to expression vectors designed to express the desiredprotein such that upon selection for the selectable marker, one alsoselects for expression of the desired protein. However because aftersuch selection, the resulting titres of the desired protein aretypically not sufficiently high, the selected cells are also subjectedto ‘amplification’ regimes. These regimes normally involve subjectingthe cells to certain toxic drugs that inhibit the selectable marker.Through such inhibition, populations of cells will be selected that haveincreased expression levels of this marker. Often this leads toincreased expression levels of the operatively linked expressioncassettes as well. Such increased expression or ‘amplification’ normallyoccurs due to genomic rearrangements resulting in increased copy numberof selectable marker and operatively linked expression cassettes. Oftenthrough such ‘co-amplification’, titres are sufficiently improved toemploy the resulting best clones to produce suitably high levels of thedesired protein or proteins. When the vector copy number in individualcells subjected to amplification regimes have been further investigated,it has been observed that up until a ‘plateau’ of protein production isapproached, the levels of production observed are typicallyproportionate to the increase in gene copy number (Bebbington andHentshcel ibid).

Many different selectable markers suitable for amplification and sotermed amplifiable selection markers have been identified to date. Eachidentified also has an associated ‘selection and amplification’ agentadded to the cell culture media during selection and amplificationregimes. Examples of such selectable marker/agent combinations include:adenosine deaminase/deoxycoformycin, aspartate transcarbamylase/N(phosphoacetyl)-L-aspartate, dihydrofolate reductase/methotrexate,glutamine synthetase/methionine sulphoximine, metallthionein-I/heavymetal, multi-drug resistance/adriamycin (see Bebbington and Hentschelibid, Kellems 1991; Current Opinion in Biotechnology 2: pp 723-729).Additionally, it is more recently reported that antibiotic selectionmarkers such as those conferring resistance to neomycin/G418 and zeocincan also be sometimes employed to increase copy number and so onoccasion have been used as selection and amplification markers whencombined with the appropriate cognate selection and amplification(antibiotic-based) selectable agent (e.g. Sauttle and Enenkel: BiotechBioeng 2004 89 pp 530-538, and Kwaks et al: Nature Biotech 2003; 21; pp553-558)

Whilst there are a number of methods to select the best geneticallyengineered cells for this purpose, the two most commonly used selectionpressures are the glutamine synthetase (GS) and dihydrofolate reductase(DHFR) based selection methods.

The GS method involves operatively linking a glutamine synthetaseexpression cassette to that of the therapeutic protein expressioncassette or cassettes. The subsequent operatively linked vectors aredelivered to cells and vector chromosomal integration is selected for bydepletion or withdrawal of glutamine from the media in which the cellsare cultured. Addition of the glutamine synthetase inhibitors such asmethionine sulfoximine (MSX) is often added to the culture media inorder to ensure glutamine synthetase activity above and beyond that ofendogenous host cell levels is selected for. The alternative DHFRselection method involves operatively linking a DHFR selection pressureto that of the therapeutic protein expression cassette or cassettes. Theoperatively linked vectors are delivered to cells and vector chromosomalintegration selected for by withdrawal or depletion of nucleosides (e.g.hypoxanthine and thymidine). Typically for the DHFR method, it iscommonplace to employ DHFR-negative host strains such as CHO DG44 or CHODUX-B11. It is also commonplace to employ selection and amplificationagents such as methotrexate (MTX).

The addition or stepwise titration of increasing amounts of the MSX orMTX selection and amplification agents in the respective GS and DHFRselection systems is often undertaken in order to augment expression byincreasing gene copy number. Such methods can involve the addition ofthe selection and amplification agent to the cell culture directly.Alternatively the agent can be added to the growth media prior to themedia being used in such cell culture. This addition or titration ofsuch agents direct to either cell cultures, or media then used for cellculture is typically termed ‘amplification’. For example in the GSsystem, MSX levels can be added or increased up to and beyond 500 μMwhilst for the DHFR system, MTX antifolate levels can be added orincreased up to and beyond 1 μM concentration levels. By use of suchagents in this way followed by a culture period to allow the selectionof cells that grow in the new concentration of selection agent, (eachconcentration step being termed a “round” of amplification), it has beenshown that the area of the genome harbouring the selection pressure canalso amplify thereby increasing the copy number of the selectablemarker. Consequently when the selectable marker is operatively linked tothe therapeutic protein expression cassettes, these cassettes may alsoamplify. By the use of appropriate selection and amplification agentswhen using the GS and DHFR selection system, yields of desired proteinscan be significantly improved up until a ‘production plateau’ isapproached (see Bebbington and Hentschel (ibid)). As a consequence, theclones that grow through such selection and amplification are thenscreened on titre/yield and the best clones are selected and furtherevaluated. From such titration and screening it is typical to identifyand then commit to one clone for subsequent production of the desiredprotein or proteins.

Typically both the number of ‘rounds’ of amplification and theconcentration of selection and amplification agent employed are not setor fixed in selection and amplification protocols. Instead it is typicalfor selection and amplification regimes to become progressivelystringent up to a point in which a production threshold or plateau isapproached. Specifically, when expressing antibodies, we and others haveobserved that clones approaching this plateau produce final titres incurrent extended unfed batch culture models and production bioreactorsin the range of 0.3 g to 1.5 g per litre. This typically translates intocell productivities (Qp) in the range of 10-100 pg/cell/per day duringsuch unfed batch culture conditions. However it is well known thatwhilst Qp (in pg/cell/per day terms) is important, it is not anexclusive determinant of productivity as clones with the highest Qp donot always give rise to the highest volumetric titres. For a recentreview see Wurm 2004; Nature Biotechnology Vol 22; pp 1393-1398.

Selection and amplification methods have been employed successfully togenerate cell lines used in manufacturing campaigns to make desiredproteins used in clinical trials. However whilst the titres generated byselection and amplification methodology are sufficient, such methods arestill undesirable for a number of reasons including time, cost andsafety. For example, the titration of amplification and selection agentsin cell line ‘amplification’ protocols delays clone selection and colonyoutgrowth, each round of amplification taking a month or more tocomplete. Second, selection and amplification agents like methotrexateand methionine sulfoximine are toxic chemicals which must be removed ifit is to be used therapeutically. Third, selection and amplificationagent resistance can occur in mammalian cells which can result in lessstringent selection pressure and result in clonal and product yieldinstability. Fourth, amplification can occasionally occur episomally.Such episomes and any operatively linked functional expression cassettesare not always inherited equally during cell division leading toincreased variation and instability in culture. Fifth, the genomerearrangements generated during amplification protocols can result insignificant changes to the host cell genome leading to variablephenotypes in resulting clones. Sixth, selection and amplification agentby-products, such as polyglutamated methotrexate can inhibit additionalfunctions of the cells (e.g. Allegra et al 1985 J Biological Chem 260;17 pp 9720-9726). Seventh, many of these selection and amplificationagents are also potentially toxic to the operators involved withculturing cells and running the bioreactors if they are exposed to highlevels. Eighth, it has also been observed that increasing the copynumber of integrated expression vectors in mammalian host cells canresult in increased repeat-induced-gene-silencing (RIGS) activity by thehost cell which can ultimately result in a reduction in expressionlevels from each of the integrated expression vectors (eg see McBurney MW et al Exp Cell Res 2002 274:1-8).

Consequently, it would be highly desirable to employ the selection andamplification methodology with reduced levels of selection andamplification agent in a reduced number of rounds of amplificationwhilst still achieving the same final yield of the therapeutic protein,such that the time taken to generate the final cell line is fasterand/or the level of the undesirable toxic agent needed to generate thefinal line is reduced or entirely excluded during cell line generation,selection and culture (M. Celina de la Cruz Edmonds et al (MolBiotechnology 2006 34:179-190).

With 64 triple base-pair codon combinations but only 20 amino acids, ithas been known for many decades that there is redundancy in the geneticcode. However, the use of codon bias to augment expression was notrealised until the 1980s. For example, in 1982 Bennetzen and Hall (JBiol Chem 257 pp 3026-3031) observed species specific codon bias instrongly expressed genes of both prokaryotes and eukaryotes. They alsonoted that this bias was taxonomically divergent. As a consequence itwas soon realised that one could modify the codon usage of open readingframes such to increase expression in recombinant expression systems.For example Kotula and Curtis (Biotechnolgy NY (1991) 9: 1386-9))achieved significantly improved expression of a mammalian antibody lightchain in yeast by codon adaptation of the open reading frame such tobias the codon usage towards those codons preferred by highly expressedendogenous yeast genes. Another very notable example was the codonadaptation of the green fluorescent protein to improve expression inmammalian cells (Zolotukhin S J Virol (1996) 70: 4646-54 and Yang et alNucleic Acids Res 1996 24:4592-3).

Recent data suggest that by raising the codon adaptation index (CAI)score of open reading frames encoding antibody heavy and light chains,one can marginally improve production yields in mammalian host cellswhen the resulting adapted expression cassettes are operatively linkedto the glutamine synthetase selectable markers and when the cells areincubated with the MSX selection and amplification agent. This data(presented at the IBC 2005 Cell Line Development and EngineeringConference), suggested that whilst mean expression levels were notsignificantly improved, the median positive clone in a group didincrease marginally (from 37.8 μg/ml to 51.3 μg/ml) but only when boththe heavy and light chain open reading frames were codon adapted. Morerecently M. Celina de la Cruz Edmonds et al (ibid), also recognised thedesire to reduce the levels of selection and amplification agent whengenerating engineered cell lines expressing desirable proteins with aidof selection and amplification regimes. They demonstrated that throughmodification of the seeding density of transfected cells, one can reducethe levels of MSX employed, and reduce the number of weeks required togenerate and maintain genetically engineered cell lines expressingequivalent or greater levels of the desired protein.

Recent published work has investigated the approaches of codonoptimisation combined with the use of the glutamine synthetaseselectable marker. For example the work presented by Kawley et al(Molecular Biotechnology 2006 Vol 34; pp 151-156), evaluates the impactof codon adaptation on the subsequent expression levels generatedhowever, the results reported suggest only minor improvements inexpression levels achieved.

Even more recently Carton et al (Protein Expression and Purification 55(2007) pp 279-286) also investigated the impact of codon optimisation.Their work involved part codon optimisation of heavy and light chainantibodies open reading frames by various approaches. These modifiedcoding sequences were then expressed in myeloma cells as mini-geneformats (ie containing introns) in expression cassettes operativelylinked to the gpt selectable marker. No amplification approaches werediscussed.

There is a need in the art to reduce the levels of selection andamplification agents required when employing expression systemsoperatively linked to amplifiable selection markers.

STATEMENT OF INVENTION

The present invention provides methods of reducing the levels of atitratable selectable pressure required, the number of amplificationcycles, and the time taken to generate protein expressing cell lines byaltering the codons of the desired open-reading-frames. Through the useof codon adaptation for this purpose the methods of the inventionconsistently provide sufficient yields in faster time frames saving manyweeks in cell line development activities. Furthermore the methods ofthe invention also generate cell lines with lower concentrations ofselection and amplification agent than previously achievable.Accordingly lower levels of selection and amplification marker in thefinal cells lines are observed.

The present invention provides methods to produce a cell line producinga therapeutic protein comprising the steps of:

a) obtaining a first polynucleotide sequence that encodes saidtherapeutic protein,

b) altering the first polynucleotide sequence to obtain a secondpolynucleotide sequence, wherein the codon adaptation index of thesecond polynucleotide sequence is greater than that of the firstpolynucleotide sequence and the first polynucleotide and secondpolynucleotide encode the same therapeutic protein.c) transforming at least one cell with the second polynucleotidesequence of step (b) and a third polynucleotide sequence that encodes aselection marker which is capable of providing amplification of thesecond polynucleotide sequence within said cell,d) growing said at least one cell of step (c) to create a first cellline comprising a plurality of cells, in medium that contains aconcentration of a selection agent that inhibits the growth of cells insaid cell line which express insufficient levels of the selection markerencoded by the third polynucleotide of step (c), such that the plateauof production of the protein encoded by the second polynucleotide isreached with fewer rounds of amplification and/or is reached at a lowerconcentration of selection agent than would be necessary to reach anequivalent plateau of production of said protein produced in a cell linetransformed with the first polynucleotide.

In all the comparative methods as herein described unless statedotherwise all other parameters such as amplification protocols orconcentrations of selection agents remain constant.

In one embodiment of the present invention the first cell line iscultured in bioreactors and the therapeutic protein produced ispurified.

In one embodiment of the present invention the codon adaptation index ofthe second polynucleotide sequence is greater than 0.9, in a furtherembodiment the codon adaptation index of the second polynucleotidesequence is greater than 0.91, in yet a further embodiment the codonadaptation index of the second polynucleotide sequence is greater than0.92, in yet a further embodiment the codon adaptation index of thesecond polynucleotide sequence is greater than 0.95.

In another embodiment of the present invention the level of selectiveagent required to achieve the arithmetic equivalency of therapeuticprotein production yield is reduced to less than 50% when compared tothe amount of selective agent used for the same method using the firstpolynucleotide sequence. In a further embodiment the level of selectiveagent is reduced to less than 25% when compared to the amount ofselective agent used for the same method using the first polynucleotidesequence, in yet a further embodiment the level of selective agent isreduced to less than 5% when compared to the amount of selective agentused for the same method using the first polynucleotide sequence in yeta further embodiment the level of selective agent is reduced to lessthan 3% when compared to the amount of selective agent used for the samemethod using the first polynucleotide sequence.

In one embodiment of the present invention there is provided a method toproduce a cell line producing a therapeutic protein comprising the stepsof:

a) obtaining a first polynucleotide sequence that encodes a therapeuticprotein and which possesses a codon adaptation index score of less than0.9.

b) obtaining a second polynucleotide sequence that encodes a therapeuticprotein wherein the codon adaptation index of the polynucleotidesequence is greater than 0.9.

c) transforming a cell line with the second polynucleotide sequence thatencodes the therapeutic protein and a third polynucleotide sequence thatencodes a selection marker which is capable of providing amplificationof the second polynucleotide,

d) growing said at least one cell of step (c) to create a first cellline comprising a plurality of cells, in medium that contains aconcentration of a selection agent that inhibits the growth of cells insaid cell line which express insufficient levels of the selection markerencoded by the third polynucleotide of step (c), such that the plateauof production of the protein encoded by the second polynucleotide isreached with fewer rounds of amplification and/or is reached at a lowerconcentration of selection agent than would be necessary to reach anequivalent plateau of production of said protein produced in a cell linetransformed with the first polynucleotide.

In one embodiment of the present invention the cell line to betransformed is metabolically deficient due to disruption or inhibitionof an endogenous cellular enzyme.

In a further embodiment of the present invention the cell line to betransformed is deficient in a nucleoside synthesis pathway.

In one embodiment of the present invention the therapeutic protein is anantibody, a derivative thereof or an antigen binding fragment.

In one embodiment of the present invention the therapeutic protein is amonoclonal antibody.

In one embodiment of the present invention the selection marker is apolynucleotide encoding Dihydrofolate reductase (DHFR) and the selectionagent is an antifolate. In a further embodiment the antifolate ismethotrexate.

In another embodiment of the present invention the selection marker is apolynucleotide encoding Glutamine synthetase and the selection agent ismethionine sulfoximine.

In one embodiment of the present invention only one round ofamplification is required to achieve a plateau of protein production.

In one embodiment of the present invention the final yield oftherapeutic protein is greater than 0.3 g/L in an unfed batch, in afurther embodiment the final yield is greater than 0.5 g/L in an unfedbatch, in yet a further embodiment the final yield is greater than 0.8g/L in an unfed batch.

In another embodiment of the present invention the concentration of MTXused is less than 50 nM or less than 25 nM or less than 10 nM. In afurther embodiment of the present invention the concentration of MTXused is 5 nM

In another embodiment of the invention, only one amplification step, andso only one concentration of the selection and amplification agent, isrequired in the cell culture medium to achieve a plateau of proteinproduction in the cells that are selected in said culture medium.

In one embodiment of the present invention there is provided an antibodyproduced by the method of the invention. In a further embodiment thereis provided an antibody produced by this method wherein the antibodyproduced comprises at least one heavy chain and which has less than orequal to 5% of non-glycosylated heavy chain. In a further embodiment theantibody's heavy chain is 95% glycosylated, or is 96% glycosylated, oris 97% glycosylated, or is 98% glycosylated, or is 99% glycosylated. Inyet a further embodiment the antibody is 100% glycosylated. In oneembodiment of the present invention the highly glycosylated antibody isa monoclonal antibody. In a further embodiment the highly glycosylatedantibody is an anti-ß-amyloid antibody. In yet a further embodiment theantibody has a heavy chain sequence of SEQ ID 18 and a light chainsequence of SEQ ID NO.19.

In another embodiment of the present invention there is provided anantigen binding fragment according to the invention described hereinwherein the fragment is a Fab, Fab′, F(ab′)₂, Fv, bispecific, diabody,triabody, tetrabody, miniantibody, minibody, isolated variable heavychain region or isolated variable light chain region, serum derivedproteins (e.g. growth factors, cytokines, albumins etc) or combinatorialfusion thereof.

In another embodiment of the invention there is provided a stablytransformed host cell comprising a vector comprising one or moreexpression cassettes encoding a heavy chain and/or a light chain of theantibody or antigen binding fragment thereof as described herein. Forexample such host cells may comprise a first vector encoding the lightchain and a second vector encoding the heavy chain. Alternatively suchexpression cassettes can be combined prior to delivery.

In another embodiment of the present invention there is provided a hostcell according to the invention described herein wherein the cell iseukaryotic, for example where the cell is mammalian. Examples of suchcell lines include Chinese Hamster Ovary, BHK, HEK-293, NSO or PerC6.(for recent review see Wurm 2004: Nature Biotechnology 22; 11 pp1393-1398). Such host cells may also contain advantageous genotypicand/or phenotypic modifications e.g. the CHO-DG44 host strain has copiesof its dhfr gene disabled whilst other hosts might have the glutaminesynthetase genes disabled. Alternative modifications may be to theenzyme machinery involved in protein glycosylation (e.g., Yamane-Ohnukiet al, Biotech Bioeng 2004 87: pp 614-622, Kanda et al, Journal ofBiotechnology, 2007 130: pp 300-310, Imai-Nishiya et al BMC Biotechnol,2007 7:84). Yet others may have advantageous genotypic and/or phenotypicmodifications to host apoptosis, expression and survival pathways (e.g.Tey et al Biotechnol Bioeng 2000 68: 31-43, Yallop et al ModernBiopharmaceuticals 2005 Chapter 3 pp 779-807, Nivitchanyang et alBiotechnol Bioeng 2007 98:825-41, Figueroa et al Biotechnol Bioeng 200797:87-92). These and other modifications of the host alone or incombination, can be generated by standard techniques such asover-expression of non-host or host genes, gene knock-out approaches,gene silencing approaches (eg siRNA), or evolution and selection ofsub-strains with desired phenotypes. Such techniques are wellestablished in the art.

In another embodiment of the present invention there is provided amethod for the production of a therapeutic protein according to theinvention described herein which method comprises the step of culturinga host cell in a culture media, for example serum-free culture media.

In another embodiment of the present invention there is provided amethod according to the invention described herein wherein saidtherapeutic protein is further purified to at least 95% or greater (e.g.98% or greater) with respect to said antibody containing serum-freeculture media.

In one embodiment of the invention there is provided mammalianexpression vectors containing open reading frames that possess CAIscores above 0.9 and which encode antibodies, antibody relatedpolypeptides, or derivatives or fusions thereof.

In another embodiment there is provided a first cell line transformedwith a second polynucleotide sequence having a codon adaptation indexthat is greater than a first polynucleotide sequence wherein the firstpolynucleotide and second polynucleotide encode the same therapeuticprotein and further comprising a third polynucleotide sequence thatencodes a selection marker which is capable of providing amplificationof the first polynucleotide sequence, wherein said first cell lineproduces a higher yield of said therapeutic protein compared with asecond said cell line transformed with said first polynucleotideencoding said therapeutic protein when grown in selectable medium.

In a further embodiment there is provided a second cell line transformedwith a second polynucleotide sequence that encodes a therapeutic proteinand has a codon adaptation index that is greater than 0.9 and furthercomprising a third polynucleotide sequence that encodes a selectionmarker which is capable of providing amplification of a secondpolynucleotide sequence, wherein said second cell line produces a higheryield of said therapeutic protein compared with a first said cell linetransformed with a first polynucleotide encoding said therapeuticprotein wherein said first polynucleotide has a codon adaptation indexthat is less than 0.9 when grown in selectable medium.

In one embodiment of the present invention the CAI score of above 0.9 iscalculated using the EMBOSS CAI scoring metric as described in Table 6.

In another embodiment of the present invention there is provided a cellline comprising a vector or an expression cassette according to theprevious embodiments as described herein.

In yet a further embodiment there is provided a cell line or its progenyobtainable by the methods of the present invention.

In another embodiment of the present invention there is providedmammalian cells with genomes containing integrated or episomallymaintained open reading frames that possess CAI scores above 0.9(derived using the EMBOSS codon usage table E.human.cut) which encodefor antibodies, antibody related polypeptides or derivatives thereof.

Throughout the present specification and the accompanying claims theterm “comprising” and “comprises” incorporates “consisting of” and“consists of”. That is, “comprising” and “comprises” are intended toconvey the possible inclusion of other elements or integers notspecifically recited, where the context allows.

Throughout the present specification and the accompanying claims theterm “plateau of production” means the level of expression approached inextended unfed batch cultures whereby additional rounds of amplificationtypically produce less than a 2-fold increase relative to the parentalamplified clone. When clones are engineered specifically to produceantibodies, clones producing between 0.3 g to 1.5 g per litre instandard extended unfed production cultures can typically be consideredas approaching this plateau of production when using current unfedextended culture regimes and media recipes.

Single-cell sub-cloning of final clones approaching a production plateaucan be undertaken by many standard methods including flow sorting (e.g.depositing a cell per well in a 96-well plate), soft-agar colonypicking, or limiting dilution cloning. To ensure single-cell outgrowthin recipient wells, sometimes conditioned media or temporary feedercultures should also be employed to support growth of the depositedotherwise lone cell. If live feeder co-cultures are required, then thesecan readily comprise parental host cells without integrated selectablevectors as such host cells can then be selected against once thedeposited single cell clone begins dividing healthily.

The term open reading frame (ORF) as used throughout this specificationrefers to the nucleic acid coding sequence encoding a desiredpolypeptide chain or chains. The codons contained within such ORF codingsequences can be contiguous or alternatively they can contain introns.When included, such introns or intervening sequences are then typicallyremoved via splicing reactions in the host cell prior to formation ofthe final, contiguous open reading frame in the mature mRNA.

The “yield” as used throughout this specification refers to theconcentration of a product (e.g., heterologously expressed polypeptide)in solution (e.g., culture broth or cell-lysis mixture or buffer) and itis usually expressed as mg/L or g/L. An increase in yield may refer toan absolute or relative increase in the concentration of a productproduced under two defined set of conditions.

The term operatively linked refers to the use of selectable andamplification markers employed to select host cells containingexpression cassettes expressing desired protein products. This can beachieved by cloning the selectable and amplification marker into thesame plasmid or vector as that containing the expression cassetteexpressing the desired protein or alternatively can be delivered to thecell on a separate plasmid or vector.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: A schematic representation of the RSV promoter based vectorsused in project 2, 3, 4, 5, 6 (a), 6 (b) and 7 (a). For the EF-1 alphapromoter based evaluations (of project 6 and 7) the RSV promoter wasreplaced with a human EF-1 alpha derived promoter plus first intron (seeKim D W Gene 1990 91: 217-23). This was obtained via PCR from humangenomic DNA. This EF-1 alpha promoter was then cloned in these vectorsin place of the RSV based promoter

FIG. 2: Non-adapted heavy chain of project 5 with a CAI score of 0.809and employed in project 5 (a) SEQ ID NO 10

FIG. 3: Non-adapted light chain of project 5 with a CAI score of 0.761and employed in project 5 (a) SEQ ID NO 11

FIG. 4: Heavy Chain ORF of project 5 (b) with increased CAI score(0.847). See Table 5. See SEQ ID 12

FIG. 5: Light Chain ORF of project 5 (b) with increased CAI score(0.833). See Table 5. See SEQ ID 13

FIG. 6: Heavy Chain ORF of project 5 with increased CAI score (0.872).This sequence was employed in antibody project 5 (c). See SEQ ID NO 14.

FIG. 7: Light Chain ORF of project 5 with increased CAI score (0.894).This sequence was employed in antibody project 5 (c). See SEQ ID NO 15.

FIG. 8: Heavy Chain ORF of project 5 with increased CAI score (0.982).This sequence was employed in antibody project 5 (d). See also SEQ ID16.

FIG. 9: Light Chain ORF of project 5 with increased CAI score (0.976).This sequence was employed in antibody project 5 (d). See also SEQ ID 17

FIG. 10: Heavy-chain and light-chain RNA and protein levels for antibodyproject 5.

FIG. 11: Codon Adaptation Methodology in Detail

FIG. 12: Example Product NGHC data obtained during project 5 final cloneselection.

FIG. 13: Example titre generated from 097-7 (project 5 (d), 5 nM MTXchosen clone CAI HC 0.982, LC 0.976) with approximately 3-monthsadditional development work after final cell-line amplification andselection.

FIG. 14: Relative levels of DHFR gene copy and protein and Neo gene copyobserved in engineered cells in various projects.

FIG. 15: Depicts the average results for Top, 20^(th) and 50^(th)titres.

DETAILED DESCRIPTION

The codon usage frequency of the genes encoding the therapeuticpolypeptides produced according to the present invention is measured anddefined by codon adaptation index (CAI).

Codon adaptation is the adaptation of the codons of an open readingframe to the synonymous codons preferred in human/mammalian genes whilstavoiding the introduction of unwanted secondary sequence functions thatimpede expression of the resulting open reading frames. We have observedthat preferred human codons are also very suitable even when subsequentexpression is planned in non-human mammalian cells (e.g. hamster derivedcells). However, if the most preferred codon for any given amino aciddiffers in a given mammalian species then this can also be employedinstead of the human preference. The “CAI score” generated for each openreading frame highlights the degree the open reading frame is adapted tothe use of synonymous codons most preferred by human/mammalian genes.

Within the context of the present invention, a CAI score of 1 means thatthe most optimal codon is used for each amino acid in each codonposition. For optimal results in the methods of the present inventionthe genes encoding the therapeutic protein have a CAI which issufficiently close to 1 such that the desired level of expression of thetherapeutic protein is achieved with significantly less selection andamplification agent and/or in a faster time relative to that observedwhen expressing the naturally occurring starting sequence, for example,the CAI is at least 0.9, or at least 0.95 or at least 0.975.

It is however not necessary to replace all codons with most preferredcodons, or replace all least preferred with more preferred codons. Theonly requirement is that the resulting sequence possesses an unnaturallyhigh CAI score and does not contain expression disrupting elements.Commercially available software such as Leto 1.0 (Entelechon,Regensburg, Germany) can design a sequence of suitably high CAI score.To further help guide in designing codon adapted sequences for use inthis invention, 24044 RefSEQ database human transcript products havebeen analysed (NM_prefixed accession numbers) derived from NCBI genomebuild number 36. The CAI score range was calculated and was from 0.593to 0.894 with an average score of 0.720. The highest score (0.894) for aknown and expressed gene (rather than theoretical) in this database wasgenerated by the keratin associated protein 5-8 (KRTAP5-8; NM_021046)and by the late cornified envelope 1A (LCE1A; NM_178348). Additionally adatabase of 21182 human IgG cDNA's revealed that IgG scores range from0.576 to 0.878 with an average of 0.766. To help guide those skilled inthe art, sequences suitable for use in the invention could possess CAIscores above and beyond that of naturally occurring highest human genessuch as the late cornified envelope 1A (LCE1A; NM_178348). Morepreferably CAI scores above 0.9 should be employed.

It was observed that if codon adaptation was carried out across ashorter sequence (for example just the variable region) then anincreased level of high producing clones is observed however, when codonadaptation is carried out across the entire open reading frame then thebreadth (i.e. number) of high producing clones generated is increasedstill further (see Table 5).

Due to the sequence of preferred codons, typical adaptation approacheswill normally by default also avoid introducing high-scoring ARE (AURich Elements see Akashi et al Blood 1994; Vol 83 pp 3182-3187)) RNAinstability sequences. However occasionally after codon adaptation thereis a requirement to remove expression disrupting sequence elementsaccidentally introduced. These include but are not limited to:

(i) Functioning splice sites,

(ii) Areas of dyad symmetry (e.g. direct, inverted or palindromicsequences) that noticeably reduce expression levels and/or increaserecombination rates between the sequences.

(iii) Functioning instability sequences.

On the rare occasions when such unwanted disruptive elements are createdduring adaptation it is recommended a less preferred but not leastpreferred human codon (unless choice is limited) be employed to disruptthe local sequence to inactivate function. Small deviations from maximalscores will not significantly impact use of the resulting open readingframes in this invention.

It is also recognised that if small areas of an open reading frameremain non-adapted (e.g. to retain useful restriction sites) then thiswill not significantly impact overall CAI score.

If the open reading frame encodes for a fusion, hybrid or chimericprotein it is encouraged that the CAI score is increased in a samemanner as described above. Again this adaptation towards synonymouscodons preferred by the host cell for the expression of highly expressedendogenous genes should be undertaken for each and every component ofgene or cognate cDNA of the fused or engineered open reading frames. Forpurely synthetic coding sequences present in a protein (i.e. sequencefor which no prior sequence exists) it is advised to introduceunnaturally high human gene CAI scores in precisely the same manner.Open reading frames with CAI scores of above and beyond the latecornified envelope 1A human gene should be employed in this invention.

The present invention herein described is the first to describe a methodof reducing the levels of a selectable and amplification agent required,the time required, and the number of amplification cycles required inorder to generate genetically engineered cell lines expressing desiredlevels of protein by codon adaptation of the open-reading-framesencoding the protein. Through the use of codon adaptation for thispurpose sufficient yields are being observed in faster time frames andso saving many weeks in cell line development activities. Furthermore weare also generating equivalent or improved cell lines by using lowerconcentrations of selection and amplification agent than we have everpreviously achieved. Indeed it is likely that when such improvements asdescribed herein are combined with standard cell culture and seedingprotocol improvements as those described by Celina de la Cruz Edmonds etal (ibid), further reductions in the levels of selection andamplification agents, and further reduction in the time needed togenerate equivalent or improved yields from genetically engineered celllines, will be observed.

The present invention is suitable for use when the therapeutic proteinis a glycoprotein. Whilst previous work discloses the fact that one cancontrol protein product glycosylation by modification of processduration, temperature, pH, osmolarity and media constituents andadditives etc (e.g. see WO2002076578 and references therein), we havefound that codon adaptation of open reading frames encoding therapeuticpolypeptide sequences (in the case of antibody therapeutic polypeptides)is able to decrease levels of incomplete glycosylation and levels ofreduced site occupancy independently of CHO cell sub-type, selection andamplification regimes or media culture conditions.

This surprising observation is the first to demonstrate that one canimpact protein glycosylation profile via open reading frame codonadaptation. By employing the codon-adaptation approaches as describedherein, a robust manufacturing process can thus be ensured which dependson the sequence of the gene rather than the conditions that the hostcell is grown in. In turn this allows for greater opportunity to improveculture conditions and feed regimes through traditional media and feeddevelopment iterations without excessive concern over the resultingimpact on the product glycoprofile.

The degree of codon adaptation can be measured using the method firstdescribed by Sharp and Li (Nucleic Acid Res 1987 15:1281-95). Sharp andLi proposed the Codon Adaptation Index (CAI) score which is essentiallyderived from the codon preference statistics, but normalized for eachamino acid so as to exclude the effects of variation in amino acidcomposition between different genes. This CAI metric is readilyavailable (e.g. via EMBOSS The European Molecular Biology Open SoftwareSuite (see Rice et al 2000: Trends in Genetics 16; pp 276-277)).

In order to score open reading frames intended for use in thisinvention, one must first use the appropriate reference database. Firstone should consider the cell host to be used then one should identify areference table of relative synonymous codon usage (RSCU) for expressedgenes in said cell host. Typically human RSCU databases are suitable forreference when expressing resulting open reading frame in any mammaliancell type. One example of a database is that provided by EMBOSS whichuses as a reference the Ehum.cut codon usage table to determine codonusage preferences in human cells. An alternative reference codon usagetable is that described by Massaer et al (ibid) in which a smallernumber of highly expressed human genes are employed to determine codonpreference. Whilst these two reference tables broadly agree on the mostpreferred codon, there is one notable divergence for one amino acid(arginine). Therefore when designing open reading frames for use in thisinvention it is logical to cross reference the same codon usage tablesto (i) determine the most preferred codons to include in the openreading frame and then (ii) CAI score the open reading framesubsequently generated to ensure the score is sufficiently high to besuitable for use in this invention. For example if the Massaer et aldatabase is employed regularly to design open reading frames forexpression in human and mammalian cells and therefore CGC codon isconsidered most preferred to encode arginine then it is logical to alsouse this preference reference data when determining the CAI score of theresulting open reading frames generated.

The methodology as described herein is particularly suitable whenexpressing antibodies or derivatives thereof and is particularlyeffective when combined with expression cassettes driven by promoter andexpression elements derived from the EF-1 alpha gene. Expressioncassettes driven by other promoter and expression elements (e.g. derivedfrom the RSV LTR) are also suitable. It is well known in the art thatexpression cassette elements (for example promoters, enhancers, matrixattachment regions (MARS), insulators, untranslated regions, interveningsequences such as introns and polyadenylations sites) can be combined inmany different combinations to create suitable expression cassettes todrive expression of the desired open reading frames and to driveexpression of the selection and amplification markers employed in thisinvention.

Once clones in any given cell line development protocol approach aproduction plateau in unfed extended batch production, it is observedthat additional laboratory activities are best focused on methodologiessuch as (i) single cell cloning of the best clones, (ii) fed-batchprocess development, (iii) perfusion style process developments, (iv)bespoke media and feed recipes and regimes and (v) further cultureadaptation. For example once a production threshold is reached for anindividual clone, its derived single-cell sub-clones are normally morestable and high-yielding than amplified daughter clones generated byfurther selection and amplification regimes in yet more stringent levelsof selection and amplification agent. Indeed increasing selection andamplification of final clones already approaching a threshold ofproduction often leads to instability and after initial improvements,can often ultimately lead to similar or even lower titres in extendedunfed production model batch cultures than the amplified parental clone.Therefore whilst it is recognised that on occasion a rare and fortuitousfurther amplification event may increase titres above 2-fold in someinstances, once an expression threshold is approached, there are morereliable techniques that can instead be employed to increase stabletitres still further.

The present invention is exemplified by and not limited by, thefollowing examples.

EXAMPLES

In the past, DHFR selection methodology has been employed on more thanfifteen antibody projects. In all cases when using this methodology atleast two rounds of amplification and a minimum of 50 nM MTX as aselection and maintenance pressure to generate cell lines with suitableyields has been necessary. Typical results generated over this period oftime by this methodology are represented by antibodies 1, 2, 3 and 4 inTable 1. Antibody project 1 was carried out using standard methodologiesavailable at the time. Antibody projects 2-9 were carried out accordingto the materials and methods below.

The impact of improving the codon adaptation index (CAI) of open readingframes was studied.

Antibody 5 was first chosen for investigation. This study involvedexpressing the antibody product from wild-type (i.e. non codon-adapted)heavy and light chain antibody open reading frames (recorded as antibody5 (a)), heavy and light chain open reading frames with codon adaptationbroadly of the variable domain coding sequences only (recorded asantibody 5 (b) or 5 (c)) or codon adaptation of the entire heavy andlight chain open reading frames (recorded as antibody 5 (d)). Theresults of this study are presented in Tables 1-5.

Example 1, Materials and Methods

1.1 DNA Cloning and Vector Construction.

All DNA cloning was performed by established restriction enzyme basedsub-cloning and PCR assembly methodologies (see Molecular Cloning: ALaboratory Manual. Third Edition: Sambrook et al (CSH LaboratoryPress)). Schematic representations of the expression and selectionvectors are shown (see FIG. 1). Vectors shown exemplify the RSV promoterhowever, different promoters were used according to table 1. In allother respects the vectors remained unchanged.

1.2 Codon Adaptation.

In projects where CAI adapted ORF sequences were investigated, thesewere generated using desired overlapping oligonucleotides combined withthe aid of standard fusion polymerase chain reaction (PCR) prior tocloning and sequence confirmation; all by standard methodology (seeMolecular Cloning: A Laboratory Manual. Third Edition: Sambrook et al(CSH Laboratory Press) and Stemmer et al., Gene. 164(1):49-53, 1995).The sequences of the adapted regions of the ORFs of project 5 (b) and 5(c) were designed using the Massaer Codon Usage preference forhuman/mammalian cells (see FIG. 11).

For antibody project 5 (d), the codon adapted ORF sequences weredesigned and generated by contract service provider 1. The resultingORFs possessed a CAI score of >0.9. The codon adapted sequences encodingthe antibody variable domains for antibody project 6 were designed andgenerated by contract service provider 2. These variable domains werethen combined with the codon adapted constant domains encoding sequencesof project 5 (d) by standard sub-cloning with aid of unique cloningsites located between constant and variable regions (using SpeI forheavy chain, BsiWI for light chain) (projects 6 (b) and 6 (d)). Theresulting ORFs encoding the full length antibody for project 6 (b) and 6(d) each possessed a CAI score of >0.9. The entire codon adapted ORFs ofantibody project 7 were designed and made by contract service provider 2and the resulting ORFs possessed a CAI score of >0.9. The ORFs ofprojects 8 and 9 employed the Leto software algorithm to design thevariable domain sequence. In-frame full-length open-reading-frames werethen generated by combining these sequences with appropriate constantdomain encoding sequences (again using the SpeI and BsiWI sites asabove): For antibody 8, the sequences encoding the variable domains werefused with the respective constant domain encoding sequences fromproject 7. For antibody 9, the variable domain encoding sequencesgenerated were fused with the respective constant domain encodingsequences from project 6 (d). Once more the resulting ORFs encoding theentire heavy and light chains for project 8 and 9 each possessed CAIscores >0.9.

In FIG. 11(A). The light chain sequence encoding the CDR1 of antibodyproject 5 is shown as a representative sample sequence. The amino acidsequence of this CDR is shown. An example potential auuua instability AUrich element (ARE) is shown boxed and bold (see also Akashi et al Blood83: pp 3182-3187). The arginine codon is also highlighted. First, theincreased codon adaptation method resulted in an increased CAI scoreacross the ORF. This antibody was employed in project 5 (b). As shownthis method included most preferred codons (e.g. for Tyr) but not on alloccasions (e.g. Leu). Second, the maximal CAI score employed the mostpreferred codons according to Massaer et al. This sequence was employedin antibody project 5 (c). The final sequence provided, employed themost preferred codons according to a larger database such as thatavailable on the Codon Usage Database website. This antibody sequencewas employed in project 5 (d). In FIG. 11(B) The codon preference tablesof highly expressed genes in humans adapted from Massaer et al. In FIG.11(C) The codon preference table for human genes adapted from The CodonUsage Database (www.kazusa.org.jp/codon) for Homo sapiens (comprising89533 CD's (38691091 codons).

Note for both heavy and light chain ORFs, unique Hind III (5′) and EcoR1sites (3′) were routinely employed to shuttle open reading frames intothe expression vectors.

All sequences were confirmed prior to use in transfection.

For example sequences see FIGS. 2, 3, 4, 5, 6, 7, 8 and 9 which recordthe original and adapted open reading frame sequences of project 5. Notethat in project 5 only 5 (d) was sufficiently codon adaptated on regionsof the open reading frames for the resulting CAI score to go above 0.9.

For all CAI scores reported herein, the Ehum.cut codon usage table wasused for reference (available via EMBOSS).

These scores are calculated using the Codon Adaptation Index applicationwhich employs the methodology first described by Sharp and Li (ibid).This application is part of the EMBOSS suite. Version 2.8.0 Ehum.cutcodon usage files and the default parameter settings were used todetermine the CAI scores of the sequences.

TABLE 6 Ehum.cut codon usage table derived from EMBOSS.Column A: Codon sequence; Column B: amino acidencoded; Column C: Proportion of usage of a givencodon among its redundant set; Column D: Numberof codons per 1000 codons; Column E: Number oftimes codon observed in data set used to derive the table. (A) (B) (C)(D) (E) GCG A 0.100 6.950 10994 GCA A 0.220 15.370 24296 GCT A 0.27018.750 29645 GCC A 0.410 28.340 44818 TGT C 0.440 9.970 15764 TGC C0.560 12.630 19971 GAT D 0.460 22.530 35629 GAC D 0.540 26.940 42601 GAAE 0.420 29.040 45923 GAG E 0.580 40.670 64302 TTT F 0.450 16.640 26304TTC F 0.550 20.620 32611 GGT G 0.170 11.880 18792 GGG G 0.240 16.52026128 GGA G 0.250 17.710 28000 GGC G 0.340 23.940 37852 CAT H 0.4009.660 15276 CAC H 0.600 14.350 22687 ATA I 0.150 6.920 10941 ATT I 0.35016.280 25738 ATC I 0.500 23.380 36976 AAA K 0.410 24.120 38145 AAG K0.590 34.370 54344 CTA L 0.070 6.320 9990 TTA L 0.070 6.400 10123 TTG L0.120 11.520 18218 CTT L 0.130 11.740 18564 CTC L 0.200 18.690 29552 CTGL 0.420 38.790 61342 ATG M 1.000 22.230 35143 AAT N 0.450 17.340 27422AAC N 0.550 21.190 33512 CCG P 0.110 6.700 10588 CCA P 0.280 16.81026574 CCT P 0.280 16.970 26837 CCC P 0.330 19.900 31463 CAA Q 0.26011.930 18863 CAG Q 0.740 33.220 52535 CGT R 0.090 4.770 7535 CGA R 0.1106.040 9547 CGC R 0.200 10.750 17002 AGG R 0.200 10.780 17049 AGA R 0.20010.820 17104 CGG R 0.200 10.830 17126 TCG S 0.060 4.390 6942 TCA S 0.14011.070 17497 AGT S 0.150 11.180 17681 TCT S 0.180 14.120 22320 TCC S0.230 17.320 27389 AGC S 0.250 18.890 29874 ACG T 0.120 6.550 10364 ACTT 0.240 13.250 20954 ACA T 0.270 15.220 24071 ACC T 0.370 20.980 33176GTA V 0.110 6.920 10939 GTT V 0.170 10.880 17196 GTC V 0.250 15.44024415 GTG V 0.470 29.080 45989 TGG W 1.000 12.430 19658 TAT Y 0.43012.320 19479 TAC Y 0.570 16.510 261101.3 Cell Culture.

Suspension-adapted CHO DG44 cells were routinely passaged inanimal-derived-component-free-media to which they had been previouslyadapted. This media consisted of a basal formulation containing aminoacids, trace elements, vitamins, glucose, and yeast hydrolysate. Thismedia was also supplemented with recombinant insulin, lipids andnucleosides. Sodium bicarbonate was added to media as a buffer. Manyequivalent animal derived component free media recipes are known in theart. Initial selection for vector transformed cells was undertaken bynucleoside withdrawal (for DHFR selection) and G418 addition (forneomycin phosphotransferase selection). For titre ranking, the 96-wellassay titres were prone to variation induced by cell growth, seedingnumbers, media dispensing volumes, and evaporation kinetics across aplate. As a consequence the titres generated in shake flasks productionmodels were more indicative of cell line rank order in high-yielding,amplified cell lines. For such models all cells were seeded at the sameinitial density. In such models, viability and growth were alsomonitored.

1.4 DNA Preparation Before Transfection

Equal amounts (15 μg) of the heavy-chain and light-chain expressionvector were linearised to completion (with Not I) in a 200 μl volumeeppendorf reaction and then ethanol/sodium acetate precipitated. Thepellet was then washed in 70% ethanol, air dried and re-suspended in 50μl of molecular-biology grade water.

1.5 Preparation of CHO DG44 Cells Before Transfection

1.2×10⁷ cells (per transfection) of healthy growing cells were spun(1000 rpm for 2-10 minutes) in a 15 or 50 ml tube, washed in 15 ml ofice-cold PBS/sucrose, spun again and then re-suspended in 800 μl ofice-cold PBS sucrose. This cell suspension was then added to thepreviously prepared DNA and left on ice for 15-minutes before beingtransferred to a chilled electroporation cuvette.

1.6 Electroporation

The cuvette containing the prepared DNA and cells was electroporated ina Gene Pulser set to 25 μF and 0.38 kV and then returned to ice for 10minutes. The cells were then removed and added to 240-mls ofnon-selective media and then plated in non-selective media in 40×96-welldish at 2-5×10³ cells per well (i.e. 50 μL per well). The plates werethen wrapped in foil and incubated at 37° C. and 5% CO₂ for 48 h.

1.7 Selection, Amplification and Clone Identification

48-hours after electroporation, 150 μL of selective media was added toeach well. This selective media contains G418 and no nucleosides. Once aweek thereafter, 140 μl of media was carefully exchanged for freshselective media without disturbing the settled cell layer and after 3-4weeks, all growing clones (typically growth of 0.1 colony per well; i.e.growth in 10 wells per 96-well plate) were titred for antibodyproduction. The top ranking clones (typically 20-100) identified werethen scaled-up in the same selective media through 24-well dishes and upto 6-well dishes. These clones were then plated at 1000 cells/per wellin a 96-well dish (96-wells per clone) and then selected on selectivemedia also containing 5 nM methotrexate in a volume of 200 μl per well.After additional two to three weeks incubation, the best clones wereagain scaled up and then re-plated at 1000 cells per well but in 50 nMMTX. These clones were also screened in 96-well plates after 2-3 weeksof growth and the best scaled up and then plated at 1000 cells per wellin 96-well dishes but with 150 nM MTX. In order to evaluate final clonesfor production potential, the best clones at 150 nM MTX were thenscaled-up and evaluated in shake flask production models for titre andquality of the product generated. The best clone for project 5 (a) was aclone labelled 17-9-6-1. This generated 0.3 g per litre end-titre inunfed production models.

NB. Levels of methotrexate and the number of rounds of amplificationrequired in step 1.7 varied depending on the project and whether thesequences were codon adapted.

1.8 Titre Analysis.

For media samples obtained from 96-well plates, antibody titre wasdetermined by automated 96-well sandwich ELISA style methodology on anIGEN M-Series M8/384 analyser (Bioveris, MaryLand, USA) withmanufacturer's recommendations and standard methodologies. The sandwichconsisted of Streptavidin coated magnetic coated beads,Biotinylated-Protein A and Ruthenium labelled F(ab)2 fragments. Thesignal generated for the test sample was then compared to a serialdilution of the antibody reference standard. Whilst a highly sensitiveassay, due to assay variation combined with cell growth variables at96-well cultures, assay intermediate precision and reproducibility isrelatively low for this assay for high-yielding, amplified cell lines.For media samples obtained during shake flask and bioreactor productionmodelling, antibody titre was measured with the aid of a nephelometricmethod where a light signal is scattered by the insolubleimmune-precipitin in the reaction solution using a Beckman Coulter Imagesystem (Buckinghamshire, England) and manufacturer's recommendations andstandard methodologies. The signal generated for the test sample againbeing compared to a serial dilution of the antibody reference standard.All titres reported are approximate.

1.9 Bioreactor Shake Flask Models (Extended Unfed Batch ProductionModels).

Typically cells were seeded in standard 250 ml tissue-cultureshake-flasks at 800,000 cells per ml with vented lids containinganimal-derived-component-free media and to total volume of 120 mls.These flasks were then incubated with agitation in carbon dioxideenriched air and set temperatures to encourage and sustain cell growth.Various conditions were tested for each clone—for example at varioustemperature conditions. In the results reported herein the highest titrefor each clone (across standard conditions) tested is exemplified.Typically the production model end point titres as reported herein wererecorded at the point at which cell viability drops to approximately 50%as determined by trypan blue exclusion based assay on a Vi-Cell(Beckman) using standard Vi-Cell CHO parameter settings andmanufacturer's recommended protocol. Typically this end-point titre isgenerated after 10-20 days incubation.

1.10 Bioreactor Culture Methodology.

Standard bioreactor culturing methodologies and equipment were employedat all times. Typically to generate a seed train, cells were scaled upinto larger volumes and passaged twice a week on a repeated 3-day then4-day regime. For the work shown in FIG. 13, seed cells were then usedto inoculate 3-litre Applikon bench top bioreactors (2-litre workingvolume) run under the following process conditions: Temperature 34²C, pHset point 6.95, DO set point 30%. As with Shake flask models, cultureswere extended until cell viability dropped to approximately 50%. Thesebioreactors broadly mimic end-point titre of both shake-flask as well aslarger bioreactors used to supply clinical trial material etc.

1.11 RT-QPCR Analysis (for Results, See FIG. 10).

CHO RNA extractions and RT-QPCR reactions were undertaken by automatedsilica based extraction using the MagNA Pure and the RNA HighPerformance RNA Isolation kit and protocols (Roche). Following reversetranscription using random hexamers, the PCR reaction was undertakenusing an ABI-7700 (Applied Biosystems) and analysed via the ΔΔCtrelative quantitation algorithm using standard methodology. Thereactions were multiplexed (18S+ Target gene [heavy chain/light chain]),18S being the most abundant target was primer limited to preventinhibition of the target reactions. Probes and flanking primer pairsemployed for Q-PCR were used according to SEQ ID. NO's 1-9

Note that the heavy chain and light chain probes/primers above were notsuitable for use with project 5 (d) due to increased ORF codonadaptation undertaken in this project hence their exclusion from FIG. 10(A).

1.12 Western Blot Analysis (for Results See FIG. 10).

Standard methodology was employed and is described in detail elsewhere(e.g. see Sambrook et al IBID). In brief polyclonal equivalent cellextracts were made using whole cell lysis and protein extraction buffer.Equal amounts of each extract were then heat incubated with Laemmliloading buffer and then loaded and run on SDS-Page gels withtris-glycine running buffer to separate the protein fractions. Onceseparated, the proteins were then electro transfer blotted ontonitrocellulose membranes and then probed with a whole anti-human IgG(HRP conjugated). A signal was generated by incubation with an HRPsubstrate and recorded with X-ray film. An additional longer exposurewas required to detect antibody light-chain product for project 5 (a).

1.13 Fluorescent Methotrexate Staining to Determine DHFR Levels inClones Producing Desired Recombinant Protein.

Each clone was cultured without methotrexate for 4-5 days prior toaddition of 10 μM Alexa-Fluor 488—Methotrexate (MolecularProbes/Invitrogen, Paisley) for 18-22 hrs at 37° C. 5% CO₂ to 700,000live cells. Stained cells were then harvested and washed with media andincubated at 37° C., 5% CO₂ for 30 mins. Harvested cells were washedagain with media and then re-suspended in media, filtered and live/deadexclusion dye Propidium Iodide (Sigma, St Louis) was added beforeanalyzing on BD FACS ARIA. Data shown in FIG. 14 (A) are of gated livecells only.

1.14 qPCR Analysis of Genomic DNA for DHFR and Neo Levels.

CHO genomic DNA extraction was performed using standard kits fromQiagen. Following DNA quantitation and normalisation using aspectrophotometer reading, the PCR reaction was undertaken using anABI-7700 (Applied Biosystems) and analysed via the ΔCt relativequantitation algorithm using standard methodology. Probes and flankingprimer pairs employed for Q-PCR were used according to SEQ ID. No's20-25. results are shown in FIG. 14 (B).

Example 2, Expression of Monoclonal Antibody Heavy and Light Chains inCHO Cells

Surprisingly whilst the high CAI scoring ORF's, but still of naturalscore (i.e scoring less than highest observed natural human ORF's suchas late-cornified envelope a! LCE1A; NM 1783480) of project 5 (c)generated a higher top titre than the unnaturally high CAI scoring openreading frames of project 5 (d), the breadth (number) of high producersin 5 (d) were improved (see Table 5). The best clones of 5 (a), (c) and(d) were then amplified and evaluated further. For this furtherevaluation, the 5 (a) clones were progressed as the control and torepresent the typical project titres observed prior to the resultsdescribed herein. The results of this work generated a high producing,stable clone (titre and growth observed for 40 passages) from project 5(d) in an unexpectedly fast time and with reduced levels ofamplification. Indeed the levels of methotrexate required to generatethe final cell line from 5 (d) was significantly lower (97% lessmethotrexate) relative to that required to generate equivalent celllines expressing similar or lower levels of the same protein productfrom non codon-adapted open-reading-frames of project 5 (a) (see Table1). Further detailed analysis was carried out—See Tables 2-4.

To investigate if the binding properties of the resulting recombinantproteins generated by modified CAI scored ORFs were impacted by codonadaptation, the binding characteristics were compared and analysed forthe antibody of project 5 encoded by either ORFs of CAI score 0.809(HC)/0.761 (LC) or by ORFs of CAI score 0.982 (HC)/0.976 (LC). Bothmaterials were generated in bioreactors and then purified by equivalentpurification regimes. Through this comparison it was shown that thebinding characteristics of the antibody were unaffected by the CAIalterations to the ORFs that encoded this antibody.

From project 5 (d) the top producing clone, 097-7, as shown in Table 1,was single-cell cloned to ensure clonality of the cell line, with theresulting titre of the best sub-clone generating a near 2-fold increasein unfed extended batch cultures relative to the non-cloned parent. Thetitres shown in FIG. 13 are generated from unfed batch cultures in twoseparate 3-litre Applikon bench top bioreactors as described in 1.10.

TABLE 1 For each protect, the final clone chosen for subsequent furtherdevelopment and banking is presented. Also highlighted are the 96-welltitres (ng/ml) generated for each final clone at each stage of its cellline development. Typical data generated prior to the results asdescribed herein are represented as antibody projects 1, 2, 3 and 4.Note that project 2 and project 4 express the same product. For project2 and 4 all activities were carried out in two independent laboratoriesusing the same vectors, host cells and protocols but different laboperators and equipment. All titres shown below at the 0, 5, 50 and 150nM MTX are those generated at 96-well stage. Higher titrations showed nosignificant improvement in batch production models and so lower MTXclone progressed (see Table 2). FIO = For information only, notrequired. Project 6 (a) was discontinued before plateau was reached dueto better titres from projects 6 (b) -6 (d). Antibody 1 2 3 4 5 (a) 5(d) 6 (a) Final Best ACC522(L4) 15-27-4 C9-13-9 129-1-3-1 17-9-6-1 O97-7141-6-4 Clone (DRC349) (best A2 96- well titre)  0 nM MTX 120 110 20 4112 340 16  5 nM MTX 530 180 150 920 32 880 72  50 nM MTX 1720 1240 4905231 310 6700 438 (FIO) 150 nM MTX Not Not 1910 23000 1670 Not Notundertaken- Required Required Required required Codon No No No No No YesNo Optimised? Promoter CMV RSV RSV RSV RSV RSV RSV CAI 0.679 0.811 0.8140.811 0.809 0.982 0.818 (Heavy Chain) CAI 0.674 0.767 0.763 0.767 0.7610.976 0.755 (Light Chain) Weeks to ~15 12 19.5 18 19 7 >15 generatefinal line in 96 well Non-Fed 0.3 g 0.3 g 0.5 g 0.9 g 0.2 g 0.7 g Notdetermined- Production (100%) (100%) (300%) (300%) (300%) (10%)estimated Bioreactor at <0.1 g Model (% MTX) (100%) Antibody 6 (b) 6 (c)6 (d) 7 (a) 7(b) 8 9 Final Best 280-9-6 58-3-3 P100-1 C65-5 74-3 454-6390-8 Clone  0 nM MTX 78 76 2350 64 2054 1280 2055  5 nM MTX 1140 11602025 5700 2125 1690 14055  50 nM MTX 4170 5775 Not Not Not Not NotRequired Required Required Required Required 150 nM MTX Not Not Not NotNot Not Not Required Required Required Required Required RequiredRequired Codon Yes No Yes Yes Yes Yes Yes Optimised? Promoter RSV EF-1aEF-1a RSV EF-1a EF-1a Ef-1A CAI 0.976 0.818 0.976 0.977 0.977 0.9540.975 (Heavy Chain) CAI 0.978 0.755 0.978 0.973 0.973 0.919 0.973 (LightChain) Weeks to 14 ~15 10 8.5 8.5 7.5 8 generate final line in 96 wellNon-Fed 0.5 g 0.9 g 0.9 g 0.4 g 0.5 g 0.6 g 2.2 g Production (100%)(100%) (10%) (10%) (10%) (10%) (10%) Bioreactor Model (% MTX)

TABLE 5 Titre comparison of non-amplified clones generated by standardtransfection and selection protocol and then selected on nucleosidewithdrawal and G418 addition. All cell lines contain the same vectorsexpressing the same antibody (of project 5) but from open reading framesencoding with differing CAI scores. For project 5 (a) (non-optimised)three further transfections were performed but are not shown as resultswere essentially background. In Figure (A) “% titre > 5 ng” refers tothe % of wells after screening recording above 5 ng/ml. “% titre > 50ng/ml” refers to the % of wellsscreened recording above 50 ng/ml. “Toptitre” refers to the highest scoring titre of all screened. “50^(th)Value” refers to the 50^(th) best titre screened. “20^(th) value” refersto the 20^(th) best titre screened. In (B), the average results for Top,20^(th) and 50^(th) titres reported in (A) are represented in histogramformat. % with % with HC CAI LC CAI titre > titre > Top 50^(th) 20^(th)Transfection SCORE SCORE 5 ng/ml 50 ng/ml titre value value 1 0.8090.761 23 0 43 6 8 2 0.809 0.761 26 0 16 5 6 3 0.809 0.761 26 0 23 6 7 40.847 0.833 43 7 376 2 17 5 0.847 0.833 62 24 778 9 76 6 0.872 0.894 4720 674 23 105 7 0.872 0.894 63 30 923 12 103 8 0.982 0.976 57 25 706 46178 9 0.982 0.976 82 61 653 80 171

TABLE 3 Detailed titre analysis in 96-well plates of the projects shownin Table 1. Titre of the best and the 50^(th) best clone is shown afterG418 addition and nucleoside withdrawal selection but with nomethotrexate addition. Promoter used to Codon drive expression of TopTitre 50th Titre Project Adaptation? antibody ORFs (ng/ml) (ng/ml) 2 NoRSV 130 2 (22nd) 3 No RSV 71 12 4 No RSV 152 22 5 (a) No RSV 43 6 5 (d)Yes RSV 653 80 6 (a) No RSV 116 14 6 (b) Yes RSV 840 89 6 (c) No EF-1a1153 87 6 (d) Yes EF-1a 2499 1426 7 (a) Yes RSV 830 50 7 (b) Yes EF-1a3467 528 8 Yes EF-1a 4090 739 9 Yes EF-1a 3108 573

TABLE 4 The top 20-100 clones as observed in projects 5 and 6 were thenscaled into 6-well dishes before being re-plated into 96-wells in mediacontaining 5 nM methotrexate. The mean and high titres of growing clonesobserved in experiments 5 and 6 selected at 5 nM (A1) and 50 nM (A2)concentrations of the methotrexate antifolate in 96-well dishes areshown below. A1 5 nm MTX A2 50 nm MTX Project Mean Titre Max Titre MeanTitre Max Titre 5 (a) 42 253 137 452 5 (d) 626 1760 1130 9500 6 (a) 134316 129 809 6 (b) 761 6340 2525 15030 6 (c) 593 2592 949 3884 6 (d) 10125075 3017 12360

TABLE 2 Examples of production plateaus: Example (A) the final chosenclone for Project 5 (d) was amplified further in 50 nM MTX. The unfedproduction models titres of parent clone (shaded) and resulting highesttitre of ‘amplified’ daughter clones are shown. Similar examples (B, C,D, E, F, G) also shown. For each example the highest titre of daughterclone after further amplification is recorded. This demonstrates thatreaching higher titres earlier is more beneficial than attempting toreach higher titres through further rounds of amplification. Production% change in Model production model 96-well end-point end-point titre MTXtitres titres after further Example Cell Line levels (ng/ml) (mg/l)amplification Example A: 097-7  5 nM 880 690 Chosen Clone 097-7-1 50 nM6700 550 −20% Project 5 (d) 097-7-3 50 nM 3300 216 −69% 097-7-5 50 nM3100 615 −11% 097-7-6 50 nM 1900 566 −18% Example B: P100-1  5 nM 2025901 Chosen Clone P100-1-8 50 nM 4570 499 −45% Project 6 (d) Example C:P100-6  5 nM 1435 468 Non-chosen clone P100-6-8 50 nM 5650 125 −73%Project 6 (d) Example D: P634-2  5 nM 3205 692 Non-chosen clone P634-2-450 nM 3685 369 −47% Project 6 (d) Example E: P502-1  5 nM 2385 357Non-chosen clone P502-1-4 50 nM 5325 157 −56% Project 6 (d) Example F:C65-5  5 nM 5700 400 Chosen Clone C65-5-7 50 nM 4515 168 −58% Project 7(a) Example G: 74-3  5 nM 2125 488 Chosen Clone 74-3-3 50 nM 820 239−51% Project 7 (b)

Example 3, Antibodies 6 and 7 and EF-1a Promoter

Codon-adaptation of the open reading frames of both the heavy and lightchains for antibody 6 was carried out, again to generate final CAIscores across the ORFs of >0.9. (See Table 1). The wild-type/startingand codon-adapted open reading frames were expressed in RSV basedpromoter expression vectors as well as a human elongation-factor-1 alpha(EF-1a) promoter based expression vector in which cis acting insulator,enhancer and promoter expression elements are instead supplied from anon-viral promoter source. The results of this work again demonstratedthat significantly less methotrexate was required to generate a finalhigh producing cell line when the open reading frame of the desiredprotein were first codon-adapted. Indeed, transfection 6 (a) in whichantibody 6 was encoded by non-adapted ORFs (ie. with CAI score of <0.9)was abandoned at 5 nM MTX stage prior to the generation of cells nearinga plateau of production. The amplification regime was not pursuedfurther in transfection 6 (a) because it was evident that significantmore resource and time would have been required to generate cell linescapable of producing equivalent yields of protein relative the yieldsalready obtained from cell lines in which ORFs of >0.9 had been employed(ie transfections 6 (b) and 6 (d). Furthermore, when comparinglike-for-like vectors plus or minus codon adaptation, it was observedthat codon-adaptation always reduced antifolate levels required. Again,for project 7, codon adaptation was carried out in a similar manner toproject 6 (see Table 1) and the codon adapted ORFs (CAI>0.9) wereexpressed in an RSV as well as in an EF-1a promoter based expressionvector. Once again, and irrespective of promoter, equivalent highyielding cell lines were generated in a faster time and with lessmethotrexate from CAI adapted ORFs when compared to all previousprojects in which non-adapted ORFs were employed to encode therecombinant products (summarised in Table 1).

Example 4, mRNA Levels

To further investigate this methodology the impact of codon adaptationon the levels of mRNA generated was investigated in like-for-likepolyclonal cell populations expressing the same product (the antibody ofproject 5) from the same vectors but from open reading frames reportingdiffering CAI scores.

CHO Cells were co-transfected with heavy chain (HC) and light chain (LC)encoding expression vectors encoding the same protein product (antibodyof project 5). Each transfected population was maintained as polyclonalpools. Each vector pair encodes the same antibody heavy chain (HC) andlight chain (LC) but from open reading frames with differing CAI scores.

The results of this experiment are captured in FIG. 10 and reveal that asignificant fold increase in mRNA levels is observed when the CAI scoreis raised for both heavy and light chain message relative to thenon-adapted controls. An equivalent increase in RNA levels (relative tostarting non adapted sequence) occurred in all adapted sequencesanalysed. Similarly, and within the limits of the western blot assay, anequivalent increase in intracellular protein levels was observed for alladapted sequences. However whilst such equivalence was observed inintracellular protein levels, there were difference in the levelssecreted. It was observed that cells containing the unnaturally high CAIscoring open reading frames generated higher polyclonal titres. Thisfurther supports the finding that the breadth of high producing clonesis improved when unnaturally high CAI scoring open reading frames areemployed in cell line development protocols.

FIG. 10

(A): Intracellular RNA levels of HC and LC message measured by RT Q-PCR:All signals normalised to ribosomal RNA and fold increases are relativeto signals generated for starting HC and LC vectors encoded by thenon-codon adapted open reading frames. Y-axis: Values range from 0 to50-fold increase in RNA signal. X axis: a (h) denotes negative controlHC signal generated from RNA extracts taken from non-transfected cells(in duplicate); b (h) denotes HC signal generated from RNA extractsderived from cells transfected with non-codon adapted HC and LCexpression vectors, as used for project 5 (a) (CAI scores of 0.809 forHC and 0.761 for LC); c (h) denotes HC signal generated from RNAextracts derived from cells transfected with codon adapted HC and LCexpression vectors, as used for project 5 (b) (CAI scores of 0.847 forHC and 0.833 for LC); d (h) denotes HC signal generated from RNAextracts derived from cells transfected with further codon adapted HCand LC expression vectors, as used for project 5 (c) (CAI scores of0.872 for the HC and 0.894 for the LC). Light chain signals generatedfrom the same RNA extracts as described above are shown as a(l), b(l),c(l) and d(l) respectively.(B): Western Blot Analysis; Equivalent cell extracts were separated bySDS-Page, blotted and interrogated with anti-product antibodies (HRPconjugated). Control of non-transfected cells is shown in lane 1.Polyclonal cells expressing product heavy and light chain open readingframes as follows; Lane 2 and 3; HC with 0.809 CAI score and LC with0.761 CAI score (protein expressed from experiment b(h) and b(l) above,vectors equivalent as those used in project 5 (a); Lane 4 and 5; HC with0.847 CAI score and LC with 0.833 CAI score (protein expressed fromexperiment c (h) and c (l) above, vectors equivalent as those used inproject 5 (b); Lanes 6 and 7; HC with 0.872 CAI score and LC with 0.894CAI score (protein expressed from experiment d (h) and d (l) above,vectors equivalent as those used in project 5 (c); Lanes 8 and 9; HCwith 0.982 CAI score and LC with 0.976 CAI score (protein expressed byvectors equivalent as those used in project 5 (d).(C); 24-hour product titres reported in ng/ml for polyclonal cellsdescribed in FIG. 10 (B).

Example 5, Different Methods to Achieve High CAI. Example (a) Project 8and Example (b) Project 9

a) For project 8 the Leto software was used to design the variabledomains. These were then fused to the identical constant domainspreviously generated for project 7 with aid of standard restrictionenzyme digest and ligation methodology. The resulting heavy chain andlight chain ORFs scored 0.954 and 0.919 respectively. These were thenemployed in a cell line development project and once again we generatedhigh yielding cell lines in a faster time frame and with lessmethotrexate than ever previously employed prior to codon adaptation(see Table 1).b) For project 9 again the Leto software was used to design the variabledomains. These were then fused to the identical constant domainspreviously generated for project 6 (d) with aid of standard restrictionenzyme digest and ligation methodology. The resulting heavy chain andlight chain ORFs scored 0.975 and 0.973 respectively. These were thenemployed in a cell line development project and once again we generatedhigh yielding cell lines in a faster time frame and with lessmethotrexate than ever previously employed prior to codon adaptation(see Table 1).

Example 6—Impact Upon Glycosylation

It was noted that the levels of non-glycosylated heavy chain (NGHC) weresignificantly lower when expressed from codon-adapted open readingframes relative to levels generated when expressed from non-adapted openreading frames even though the same host, culture media and DHFRselection and amplification system were employed for the expression inboth situations. Even more interestingly similar high levels of NGHCwere also observed when the same non-adapted open reading frames wereinstead expressed in different host cells employing in different cultureconditions and different vector, selection and amplification regime(Glutamine Synthetase/MSX) (see FIG. 12). This correlation betweencodon-adaptation of the open reading frame and reduced levels ofnon-glycosylated heavy chain reveals that through increasing the CAIscore of an open reading frame one can also improve the overall qualityof the product.

6.1 Cell Line Development with the Lonza CHOK1SV and GlutamineSynthetase Selection System (FIG. 12).

Vector construction and cell line development were undertaken accordingto the recommended Lonza (Slough) protocols. The media employed at alltimes was CD-CHO (Invitrogen). Open reading frames employed in antibodyproject 5 (a) containing non-adapted open reading frames were firstsub-cloned into the Lonza vectors pEE14.4 (for the light chain) andpEE6.4 (for the heavy chain). These vectors were then combined accordingto the recommended Lonza protocol into a single double gene vectorexpressing heavy and light chains. This vector was then delivered to theLonza suspension adapted CHOK1 strain named CHOK1SV host cells usingelectroporation as per Lonza recommended instructions and selected andamplified with glutamine withdrawal and with the addition of MSX also asrecommended. The resulting clones were titred at 96-well and the bestscaled-up into shake flasks and further evaluated. The best clone wasselected to make product in large-scale bioreactors. For further generaldetails on this approach see de La Cruz Edmonds et al 2006 MolecularBiotechnology 34:179-190)

6.2 Product NGHC Analyses

Protein was purified from culture supernatant with aid of protein Acolumns. The product was subsequently analysed with SDS capillaryelectrophoresis lab-on-a-chip equipment (Agilent Technologies, CheshireUK) under reducing conditions and according to manufacturer's protocol.The non-glycosylated heavy chain is observed as a slightly fastermigrating species relative to the main glycosylated heavy chain species(See FIG. 12B).

FIG. 12—Table (A) showing representative data from all analyses. Alsoincluded is additional work undertaken to express the non-adapted openreading frames in a different selection and amplification protocol(Glutamine synthetase/methotrexate) employing a different host cell,vector, media and culture regime (See example 6.1 above). (B) ExampleNGHC traces observed from starting non adapted ORFs versus codon-adaptedORFs. This analysis was undertaken on product purified from equivalentsized (1000-litre) bioreactors. In these representative trace overlays,harvest of bioreactors cell cultures expressing product from non-adaptedORFs (CAI: HC 0.809, LC 0.761) generated heavy chain with reduced siteoccupancy (10% non-glycosylated heavy chain) relative to productproduced from adapted ORFs (CAI: HC 0.982, LC 0.976) which containedonly 1.5% non-glycosylated heavy chain.

Example 7 Impact on Levels of Selection and Amplification Agents in theFinal Cell Lines

The addition or stepwise titration of increasing amounts of the MTXselection and amplification agent in the DHFR selection system isundertaken in order to augment expression by increasing gene copynumber. To investigate the impact of codon adaptation on the gene copynumber of the transfected plasmid DNA, two different semi-quantitativemethodologies were employed (see sections 1.13 and 1.14 for thedescription of the experiments).

Firstly FACS analysis was used. For this purpose the final cell lines orsingle cell clones thereof for projects 2, 3, 4, 5 (d), 6 (d) and 7 (b)were stained with fluorescent methotrexate and analysed by FACS. Theresults (shown in FIG. 14A) demonstrate that the levels of methotrexate,as indicated by the mean fluorescence intensity, and therefore thelevels of DHFR, correlate with the amplification level of the cell line,i.e. final cell lines selected in 5 nM MTX (projects 5 (d), 6 (d) and 7(b)) have the lowest and final cell lines selected in 150 nM MTX(project 3) have the highest DHFR levels. In addition, qPCR for DHFR andNeo was carried out on genomic DNA extracted from the final cell linesor single cell clones thereof for projects 3, 4, 5 (a), 5 (d), 7 (b) and9. The results (shown in FIG. 14B) demonstrate that lines selected in 5nM MTX (projects 5 (d), 7 (b) and 9) have significantly lower DNA levelsof DHFR and Neo—and therefore lower copy number—than lines selected in150 nM MTX (projects 3, 4 and 5 (a)).

The results discussed above demonstrate that cell lines derived from thecodon adapted ORFs (projects 5 (d), 6 (d), 7 (b) and 9 for this example)have lower gene copy number compared to lines derived from non adaptedORFs (projects 2, 3, 4, 5 (a) for this example). The use of codonadapted ORFs (CAI>0.9) therefore results in the generation of cell lines(when compared to cell lines derived from the non-adapted ORFs) withequal or higher titres, with lower levels of amplification and lowercopy number of transfected DNA. The generation of clones makingequivalent or higher levels of antibody from lower copy number, lessamplified expression vectors is highly desirable. For example it hasbeen shown that repeat-induced gene silencing (RIGS) can be induced whencopy number of an integrated expression vector is increased and thatsuch RIGS can then result in reduced expression levels from such vectorsin mammalian cells (eg see McBurney M W et al Exp Cell Res 2002274:1-8).

FIG. 14 (A). Plot of mean fluorescence observed for the final cell linefor each of projects 2, 3, 4, 5(d), 6(d) and 7(b). The staining of cellsfor DHFR was undertaken as described in materials and methods,Example 1. (B) Levels of DHFR and Neo DNA by qPCR on genomic DNA fromfinal cell lines for projects 3, 4, 5(a), 5(d), 7(b) and 9. The qPCR wasperformed as described in Materials and Methods, Example 1. To benchmarklevels observed, the lowest values (seen in Project 7 (b)) for DHFR andNeo were set to 1 then all other values were plotted as relativefold-increase above these. Also indicated below each value is whetherthe protein expressed by the cell line analysed is from an ORF withCAI>0.9 (Y=Yes and N=No) and the levels of MTX required to generate thecell line (in nM MTX).

Sequence Listing SEQ ID NO. DESCRIPTION OF SEQUENCE 1 18S RNA probe -nucleotide 2 Primer 1 3 Primer 2: 4 Heavy Chain probe: 5 Primer 1: 6Primer 2: 7 Light chain probe 8 Primer 1: 9 Primer 2: 10 Non-adaptedheavy chain of project 5 with a CAI score of 0.809 and employed inproject 5(a) 11 Non-adapted light chain of project 5 with a CAI score of0.761 and employed in project 5(a) 12 Heavy Chain ORF of project 5 withincreased CAI score (0.847): See Table 5 and FIG. 6. This sequence wasemployed in Project 5 (b) 13 Light Chain ORF of project 5 with increasedCAI score (0.833): See Table 5 and FIG. 6 This sequence was employed inproject 5 (b) 14 Heavy Chain ORF of project 5 with increased CAI score(0.872). This sequence was employed in antibody project 5 (c). 15 LightChain ORF of project 5 with increased CAI score (0.894). This sequencewas employed in antibody project 5 (c). 16 Heavy Chain ORF of project 5with increased CAI score (0.982). This sequence was employed in antibodyproject 5 (d). 17 Light Chain ORF of project 5 with increased CAI score(0.976). This sequence was employed in antibody project 5 (d). 18candidate (H2L1) heavy chain 19 candidate (H2L1) light chain 20 Primer1: 21 Primer 2: 22 DHFR Probe: 23 Primer 1: 24 Primer 2 25 Neo Probe:

SEQ ID NO. 1 5-VIC-tggctgaacgccacttgtccctctaaa-TAMRA-3′. SEQ ID NO. 25′-aggaattgacggaagggcac-3′. SEQ ID NO. 3 5′-ggacatctaagggcatcaca-3′SEQ ID NO. 4 5′-FAM-ctccggctgcccattgctctcc-TAMRA-3′. SEQ ID NO. 55′-ggaggcgtggtcttgtagttg-3′. SEQ ID NO. 6 5′-ggcttctatcccagcgacatc-3′.SEQ ID NO. 7 5′-FAM-tctcgtagtctgctttgctcagcgtca-TAMRA-3′. SEQ ID NO. 85′-cttcgcaggcgtagactttgt-3′. SEQ ID NO. 9 5′-gccctccaatcgggtaactc-3SEQ ID NO. 10 ATGGAGTTGGGGCTGTGCTGGGTTTTCCTTGTTGCTATTTTAGAAGGTGTCCAGTGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTCAGTGACAACGGAATGGCGTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATTCATTAGTAATTTGGCATATAGTATCGACTACGCAGACACTGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGTCAGCGGGACCTGGTTTGCTTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA SEQ ID NO. 11ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAATGCTCTGGGTCTCTGGATCCAGTGGGGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGAGTTAGTCAGAGCCTTTTACACAGTAATGGATACACCTATTTACATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATAAAGTTTCCAACCGATTTTCTGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCTCTCAAACTAGACATGTTCCGTACACGTTCGGCGGAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGACAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT TCAACAGGGGAGAGTGTSEQ ID NO. 12 ATGGAGCTCGGGCTGTGCTGGGTGTTCCTCGTGGCCATCCTGGAGGGAGTGCAGTGTGAGGTGCAGCTGGTGGAGAGTGGGGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGGCTGTCGTGCGCCGTGAGCGGCTTCACCTTCAGTGACAACGGCATGGCTTGGGTCAGGCAGGCCCCCGGAAAGGGGCTCGAGTGGGTGAGCTTCATCAGTAACCTGGCCTACAGTATCGACTATGCTGACACCGTGACCGGCCGCTTCACTATCTCTCGGGATAATGCTAAGAACAGCCTGTACCTCCAGATGAACAGCCTGCGCGCTGAGGACACCGCCGTGTACTACTGCGTGTCTGGAACCTGGTTCGCCTACTGGGGCCAGGGTACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA SEQ ID NO. 13ATGCGCCTGCCTGCCCAGCTGCTCGGCCTGCTGATGCTGTGGGTGTCGGGCAGCTCCGGCGACATCGTCATGACCCAGAGCCCCCTGAGTCTCCCCGTCACCCCCGGCGAACCTGCCAGCATCAGCTGCAGGGTGTCCCAGTCGCTGCTCCATTCCAACGGGTACACGTACCTGCATTGGTACCTGCAGAAGCCCGGGCAATCCCCTCAGCTGCTGATCTACAAGGTGAGCAACCGCTTCTCCGGCGTCCCGGACCGGTTCAGTGGCAGCGGCTCTGGAACCGACTTCACCCTGAAAATCAGCCGCGTGGAAGCTGAGGACGTGGGCGTCTACTACTGCAGCCAGACCCGGCATGTGCCCTACACCTTCGGCGGCGGCACAAAGGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGACAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT TCAACAGGGGAGAGTGTSEQ ID NO. 14 ATGGAGCTGGGCCTGTGCTGGGTGTTCCTGGTGGCCATCCTGGAGGGCGTGCAGTGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGTGAGCGGCTTCACCTTCAGCGACAACGGCATGGCCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCTTCATCAGCAACCTGGCCTACAGCATCGACTACGCCGACACCGTGACCGGCCGCTTCACCATCAGCCGCGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGTGAGCGGCACCTGGTTCGCCTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA SEQ ID NO. 15ATGCGCCTGCCCGCCCAGCTGCTGGGCCTGCTGATGCTGTGGGTGAGCGGCAGCAGCGGCGACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCCGGCGAGCCCGCCAGCATCAGCTGCCGCGTGAGCCAGAGCCTGCTGCACAGCAACGGCTACACCTACCTGCACTGGTACCTGCAGAAGCCCGGCCAGAGCCCCCAGCTGCTGATCTACAAGGTGAGCAACCGCTTCAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCAGCCAGACCCGCCACGTGCCCTACACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGACAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT TCAACAGGGGAGAGTGTSEQ ID NO. 16 ATGGAGCTGGGCCTGTGCTGGGTGTTCCTGGTGGCCATCCTGGAGGGCGTGCAGTGCGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCTGGCGGCAGCCTGAGACTGAGCTGTGCCGTGTCCGGCTTCACCTTCAGCGACAACGGCATGGCCTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCTTCATCAGCAACCTGGCCTACAGCATCGACTACGCCGACACCGTGACCGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGTGAGCGGCACCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGGCCGGAGCCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO. 17ATGAGACTGCCCGCCCAGCTGCTGGGCCTGCTGATGCTGTGGGTGTCCGGCAGCAGCGGCGACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCTGGCGAGCCCGCCAGCATCAGCTGTAGAGTGAGCCAGAGCCTGCTGCACAGCAACGGCTACACCTACCTGCACTGGTATCTGCAGAAGCCTGGCCAGAGCCCTCAGCTGCTGATCTACAAGGTGTCCAACCGGTTCAGCGGCGTGCCTGATAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAGGATGTGGGCGTGTACTACTGCTCCCAGACCAGACACGTGCCTTACACCTTTGGCGGCGGAACAAAGGTGGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCT TCAACCGGGGCGAGTGCSEQ ID NO 18 EVQLVESGGGLVQPGGSLRLSCAVSGFTFSDNGMAWVRQAPGKGLEWVSFISNLAYSIDYADTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVSGTWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO 19DIVMTQSPLSLPVTPGEPASISCRVSQSLLHSNGYTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQTRHVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGECSEQ ID NO 20 GAGGCAGTTCTGTTTACCAGGAA SEQ ID NO 21 CCTGCATGATCCTTGTCACAASEQ ID NO 22 Cy5-CCATGAATCAACCAGGCCACCTCAG-BBq SEQ ID NO 23GCCCGGTTCTTTTTGTCAAG SEQ ID NO 24 CTGCCTCGTCCTGCAGTTC SEQ ID NO 25Cy5-CCGACCTGTCCGGTGCCCTG-BBq

The invention claimed is:
 1. A Chinese Hamster Ovarian (CHO) cell lineproducing at least one therapeutic protein produced by a methodcomprising the steps of: (a) transforming a CHO cell line with apolynucleotide sequence comprising (i) a sequence that encodes thetherapeutic protein and has a codon adaptation index (CAI) that is equalto or greater than 0.9 as calculated using EMBOSS codon usage tableEhum.cut, and (ii) a sequence that encodes an amplifiable selectionmarker which is capable of providing amplification of the polynucleotidesequence; (b) providing at least one round of amplification in thepresence of a selection agent at a concentration of 50% or less than isrequired to achieve a cell production yield of 0.3-1.5 g/L of theheterologous therapeutic protein in extended unfed batch culture and/ora cell productivity of 10-100 pg/cell/day of the heterologoustherapeutic protein in unfed batch culture, compared with a CHO cellline with a sequence that encodes the therapeutic protein and has acodon adaptation index that is less than 0.9 as calculated using EMBOSScodon usage table Ehum.cut; and (c) selecting the cell line once thecell productivity and/or cell production yield of the therapeuticprotein is reached; wherein the CHO cell line has lower levels of theamplifiable selection and amplification marker and copy number oftransformed DNA compared to said cell line with a sequence that encodesthe therapeutic protein and has a codon adaptation index that is lessthan 0.9 and is not provided with at least one round of amplification inthe presence of a selection agent in accordance with step (b).
 2. TheCHO cell line of claim 1 comprising a vector comprising at least oneexpression cassette containing an open reading frame containing thepolynucleotide sequence that encodes a therapeutic protein with a CAIscore above 0.9, and a second expression cassette containing anamplifiable selection marker.
 3. The CHO cell line of claim 1 comprisingan amplified expression cassette containing an open reading framecontaining the polynucleotide sequence that encodes a therapeuticprotein with a CAI score above 0.9 contained within said cell line whereit is operatively linked to a second expression cassette containing anamplifiable selection marker.
 4. The CHO cell line of claim 1 whereinthe cell productivity of the therapeutic protein is in the range of10-100 pg/cell/day in an unfed batch culture.
 5. The CHO cell line ofclaim 1, wherein the therapeutic protein is an antibody with a heavychain, and the cell line produces an antibody composition comprisingless than or equal to 5% of Non-glycosylated heavy chain.
 6. The CHOcell line of claim 1, wherein the therapeutic protein is an antibodywith a heavy chain, and the cell line produces an antibody compositioncomprising at least 95% glycosylated heavy chain.
 7. The CHO cell lineof claim 1, wherein the therapeutic protein is an antibody with a heavychain, and the cell line produces an antibody composition comprising atleast 96% glycosylated heavy chain.
 8. The CHO cell line of claim 1,wherein the therapeutic protein is an antibody with a heavy chain, andthe cell line produces an antibody composition comprising at least 97%glycosylated heavy chain.
 9. The CHO cell line of claim 1, wherein thetherapeutic protein is an antibody with a heavy chain, and the cell lineproduces an antibody composition comprising at least 98% glycosylatedheavy chain.
 10. The CHO cell line of claim 1, wherein the therapeuticprotein is an antibody and the antibody is monoclonal.